Zhang Hui 2012213034

1. Determination of Hydrolyzable Tannins (Gallotannins and Ellagitannins) after Reaction with Potassium Iodate Zhang Hui 2012213034 J. Agric. Food Chem.2002,50,1785-1790.

2. Contents • Introduction • Materials and methods • Results and discussion • Conclusion

3. Introduction • Tannins hydrolyzable tannins condensed tannins • high molecular weight polyphenolics found in higher plants gallotannins ellagitannins

4. Structural formulas of representative hydrolyzable tannins from plants. Compound 1 is pentagalloyl glucose, comprised of the gallate (G,2) esters of glucose. Compound 3 is the simple ellagitannin eugeniin, comprised of both the gallate esters and a single HHDP (G−G,4) ester. Compound 5isEGCG, the gallate ester of the flavanoid epigallocatechin. Compound 6 is oenothein B, a macrocyclic hydrolyzable tannin. All four of thehydrolyzable tannins react in acidic methanol to yield methyl gallate (7)

5. Introduction • Individual hydrolyzable tannins can usually be determined by HPLC, but methods for conveniently screening large numbers of samples with variable hydrolyzable tannin compositions have not been developed. • The KIO3 method has been employed in some studies of oaks and maples. • The purpose of this work was to optimize the KIO3 method for analysis of hydrolyzable tannins.

6. Materials and methods • Reagents tannin acid，gallic acid, methyl gallate, epigallocatechin gallate (EGCG) • Methanolysis and pH Adjustment anhydrous methanol, 85 ℃, 20h,pH: 5.5±0.1 • Analysis 30 ℃ water bath， 50 min after KIO3 was added, recorded absorbance at 525 nm • HPLCAnalysis of Methanolysis Products C18 (100×4.6 mm),1.0 mL/min , ƛmax 525 nm, gradient elution

7. Results and Discussion 1 2 B A + KIO3 yellow ƛmax 525 nm Why KIO3 ？ Hypochlorite ? the lifetime of A was shorter Potassium periodate (KIO4) ? past the red intermediate to the final product

8. Disadvantages of KIO3 method different color yields in rates of reaction for structurally different gallotannins Improve Why not gallic acid？ Methyl gallate 1 2 B A + KIO3 yellow ƛmax 525 nm

9. Methanolysis Why 85℃? avoid the disappearance of some methyl gallate formation of unidentified phenolic products observed at higher temperatures Why anhydrous methanol? traces of water caused production of a mixture of gallic acid and methyl gallate

10. KIO3 Reaction How to control pH? too acidic, the chromophore A did not accumulate too basic, oxidation was accelerated and only the yellow end product Reaction of KIO3 with methyl gallate as a function of pH. Methyl gallate solutions dissolved in methanol were diluted with a series of solutions comprised of ethanolamine and ammoniumacetate buffer prepared at various pH values between 4.5 and 7.0.

11. KIO3 Reaction product A was temperature-dependent and time-dependent How to control temperature? at room temperature, there were significant day-to-day variations 30 ℃ water bath How to control time? 50 min after KIO3 was added, recorded absorbance at 525 nm

12. a linear relationship between methyl gallate and absorbance at 525 nm: Abs = 0.0132 μg + 0.0701 where R2 = 0.996, standard error of Y = 0.0303, and standard error of slope = 0.000 19.

13. Conversion of hydrolyzable tannins to methyl gallate by 20 h of methanolysis at 85°C. A Methanolysis samples (s) of tannic acid B . EGCG C. Acer leaf powder

14. Theoretical Production of Methyl Gallate from Hydrolyzable Tannins Yield of methyl gallate is calculated from molecular weights of tannin and methyl gallate and is expressed in grams of methyl gallate per gram of tannin.

15. Example:red maple leaves Analysis was done with the original KIO3 method (A) Analysis was done with the new method described (B)

16. Theoretical Production of Methyl Gallate from Hydrolyzable Tannins Yield of methyl gallate is calculated from molecular weights of tannin and methyl gallate and is expressed in grams of methyl gallate per gram of tannin.

17. Conclusion The new KIO3 method will be useful for examining hydrolyzable tannin in ecological, physiological, and nutritional studies of plants.